Optimizing Wolbachia manipulation in mosquito cell lines: This proposal focuses on Wolbachia, an obligate intracellular bacterium that is widespread in arthropods, including insects that vector diseases of humans. In infected mosquitoes, Wolbachia causes a reproductive distortion known as cytoplasmic incompatibility, which provides one of the best-known biological tools for introducing transgenic mosquitoes into field populations. Because Wolbachia can be produced only within a living cell, its implementation for control purposes requires an understanding of its growth and replication, and development of transformation technologies, within the context of the mitotic cycle of its host cell. Aim 1 describes use of mosquito cell lines that have been genetically characterized to address the hypothesis that the Wolbachia life cycle is coordinated with the mosquito cell cycle, and that Wolbachia genome replication occurs during the G1 phase of the mitotic cycle. We further hypothesize that treating Wolbachia-infected mosquito cells with chemical mutagens, specifically during the period when Wolbachia is engaged in active DNA synthesis, will enable recovery of Wolbachia mutants with selectable antibiotic resistance. Aim 2 addresses the hypothesis that cross-talk between Wolbachia and its host cell influence the outcome of infection and overall levels of Wolbachia produced. We will use proteomics-based approaches to identify proteins, and metabolic pathways, that are differentially regulated during Wolbachia infection. Proteins to be investigated include mosquito host cell proteins and Wolbachia proteins that are secreted into the host cytoplasm and regulate cell cycle progression. Pathways that are affected by Wolbachia in cell lines will be validated in mosquito reproductive tissues. Aim 3 addresses the hypothesis that Wolbachia can be transformed with DNA encoding a fluorescent protein and an antibiotic resistance gene by homologous recombination. For optimized recovery of transformed Wolbachia, a mosquito cell line will be made dependent on Wolbachia by engineering the riboflavin biosynthetic pathway. Experimental approaches will include cell cycle synchronization, manipulation of host cell nutrients and metabolic pathways, quantitative PCR, flow cytometry, protein identification by mass spectrometry, mutagenesis and selection of Wolbachia mutants, transformation of Wolbachia, and engineering of novel mosquito cell lines that depend on Wolbachia for survival. This work will advance use of Wolbachia as a gene drive mechanism amenable to direct genetic improvement for control of mosquito-borne disease.